Third observed rate of a thermodynamic cycle. (A) Four states representing ligand-unbound (A and A*) and ligand-bound (AL and A*L) conformers are linked by rate constants, where kij represents the rate of going from state i to state j. Ligand on-rates are dependent on ligand concentration, while ligand off-rates and the rates of conformational exchange are first-order. (B) Third observed rate (kobs,3) for the system in the limits of zero and infinite ligand concentrations. (C) Dependence of the third observed rate (kobs,3)on ligand concentration for three sets of rates where the rates of conformational switching are slower than the rates of binding: (1) where the rate of isomerization to A* increases upon ligand binding (---), (2) where the rate of isomerization to A decreases upon ligand binding (⋯), and (3) where the rate of isomerization to A* increases and the rate of isomerization to A decreases upon ligand binding (—).